GSM7118577: C4-2B_ENZR_H3K27ac; Homo sapiens; OTHER
100000 cells were washed twice gently with wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail). 10μL Concanavalin A coated magnetic beads (Bangs Laboratories, Cat #:BP531) were added per sample and incubated at RT for 10min. Remove unbound supernatant and resuspended bead-bound cells with dig wash buffer (20mM HEPES pH 7.5; 150mM NaCl; 0.5mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2mM EDTA) and a 1:50 dilution of primary antibody（H3K4me1: abcam,ab8895，H3K27ac: abcam, abcam，ab4729）incubated on a roating platform overnight at 4℃. The primary antibody was removed using magnet stand. Sencondary antibody （Rabbit Anti-Mouse IgG H&L：abcam，ab611709）was diluted 1:100 in dig wash buffer and cells were incubated at RT for 60 min. cells were washed using the magnet stand 2-3 times in dig wash buffer . A 1:100dilution of pA-Tn5 adapter complex was prepared in digmed buffer (0.01% Digitonin; 20mM HEPES pH 7.5; 300mM NaCl; 0.5mM Spermidine; 1× Protease inhibitor cocktail) and incubated with cells at RT for 1h. Cells were washed 2–3× for 5 min in 1 mL Dig-med buffer. Then cells were resuspended in tagmentation buffer (10mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. To amplify libraries, 21μL DNA was mixed with 2μL of a universal i5 and auniquely barcoded i7 primer. A volume of 25 μL NEBNext HiFi 2× PCR Master mix was added and mixed. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min and hold at 8 °C. libraried clean-up was performed XP beads (Beckman Counter, a63881).  The size distribution of libraries was determined by Agilent 4200 TapeStation analysis, and libraries were mixed to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer. Sequencing was performed in the Illumina Novaseq 6000 sequencer using 150bp paired-end following the manufacturer's instructions.